Real time imaging of mitochondrial ATP levels under hypoxic conditions using a FRET biosensor

Introduction

Cardiomyocytes are rich in mitochondria to meet a high demand for adenosine triphosphate (ATP) by heart, and it has been suggested that dysfunction of mitochondria and heart disease are closely related. In addition, the heart of patients with cardiac disease is susceptible to injury from hypoxia, and one of the causes of injury is oxidative phosphorylation (OXPHOS) in the mitochondria, which has a high oxygen demand for ATP synthesis, has been inhibited by hypoxia. Real-time observation of how ATP synthesis in cardiomyocytes is affected by hypoxia could be very useful in the development of for therapeutic agents for heart disease.
In this application example, we present an experiment using ATeam, a probe for detecting ATP by Förster Resonance Energy Transfer (FRET). ATeam-transfected mouse cardiomyocytes were observed in the hypoxic chamber of the CellVoyager CV7000, and changes in ATP levels in the mitochondria and cytoplasm in response to changes in oxygen levels were measured as live. We present an assay system capable of detecting mitochondria-specific decreases in ATP levels, and data on Ivermectin and Nifuroxazide, drugs that have been found in a screening using this assay to inhibit the decrease in ATP production under hypoxic conditions.

Fig. 1 Changes of FRET ratio representing ATP levels in mitochondria and cytoplasm under hypoxia.
cyto-ATeam: A Mouse cardiomyocyte cell line HL-1 stably expressing ATeam in the cytoplasm
mito-ATeam: Mouse cardiomyocyte cell line HL-1 stably expressing ATeam in mitochondria

Experiment

  1. Mouse cardiomyocyte line HL-1 stably expressing ATeam in mitochondria or cytoplasm were seeded in 96-well imaging plates (10,000 cells per well).
  2. Cells were imaged with CV7000 to detect the signal of ATeam biosensor under normal and low (1%) oxygen conditions. Image acquisition settings are as below.
    • Magnification: 60x immersion lens
    • Excitation wavelength: 445 nm
    • Emission filter: 480/17 nm for CFP, 543/22 nm for YFP
    • Imaging interval: 10-minute intervals
    • Imaging time: 5 hours
    • Oxygen standard condition: 21% O2 for 1 hour
    • Hypoxic conditions (hypoxic chamber): 1% O2 for 2 hours
  3. Using the Yokogawa Analysis Support software, the intensity of CFP and YFP fluorescence were measured.
    YFP/CFP ratio (FRET ratio) were calculated in Spotfire®. Ratiometric images were created in ImageJ.

Fig 2. Changes in ATP levels in mitochondria and cytoplasm under hypoxia. A decrease in FRET ratio indicates a decrease in ATP concentration.

Result

It was confirmed that the FRET signal indicating the mitochondrial ATP level was changed following the varying oxygen concentration in the imaging chamber, whereas the cytoplasmic ATP level did not change even under the same conditions. From these results, it was shown that live cell imaging using a FRET probe can visualize the change in ATP level with time in an intracellular site-specific manner. Thus, by using the hypoxia module of CellVoyager CV7000, the effect of hypoxia on cells can be observed and evaluated in detail in real time. Using this assay system, it became possible to screen the effect of added compounds on changes in ATP levels in hypoxia.

Figure 3. Dose-dependent effects of Ivermectin and Nifuroxazide on ATP level change in response to hypoxia stimulation.

Reference: Nagai H, Satomi T, Abiru A, Miyamoto K, Nagasawa K, Maruyama M, Yamamoto S, Kikuchi K, Fuse H, Noda M, Tsujihata Y. Antihypertrophic Effects of Small Molecules that Maintain Mitochondrial ATP Levels Under Hypoxia. EBioMedicine. 2017 Oct;24:147-158. doi: 10.1016/j.ebiom.2017.09.022. Epub 2017 Sep 19


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